Michelle G.K. Tan

Genome wide profiling of altered gene expression in the neocortex of Alzheimer's disease.

Alzheimers disease (AD) is characterized by a complex neurodegenerative process affecting multiple genes and proteins in the neocortex, many of which have not been well‐studied. In this study, we investigated genome‐wide gene alterations in the temporal cortex of a well‐characterized cohort of AD patients using a recently developed microarray platform, and compared some of the transcript changes with immunoblotting. Of the 5485 genes found to be significantly altered in AD, there were consistent patterns of changes which show that the AD transcriptome in neocortex is characterized by changes indicative of synaptic dysfunction, perturbed neurotransmission and activation of neuroinflammation. We also highlighted several genes of potential pathogenic significance which have not been well studied in AD. The current study aims to add to the growing body of knowledge relating to gene changes in AD and provide further insights into pathogenic mechanisms and potential targets of pharmacotherapy.

Selective loss of P2Y2 nucleotide receptor immunoreactivity is associated with Alzheimer’s disease neuropathology

The uridine nucleotide-activated P2Y2, P2Y4 and P2Y6 receptors are widely expressed in the brain and are involved in many CNS processes, including those which malfunction in Alzheimer’s disease (AD). However, the status of these receptors in the AD neocortex, as well as their putative roles in the pathogenesis of neuritic plaques and neurofibrillary tangles, remain unclear. In this study, we used immunoblotting to measure P2Y2, P2Y4 and P2Y6 receptors in two regions of the postmortem neocortex of neuropathologically assessed AD patients and aged controls. P2Y2 immunoreactivity was found to be selectively reduced in the AD parietal cortex, while P2Y4 and P2Y6 levels were unchanged. In contrast, all three receptors were preserved in the occipital cortex, which is known to be minimally affected by AD neuropathology. Furthermore, reductions in parietal P2Y2 immunoreactivity correlated both with neuropathologic scores and markers of synapse loss. These results provide a basis for considering P2Y2 receptor changes as a neurochemical substrate of AD, and point towards uridine nucleotide-activated P2Y receptors as novel targets for disease-modifying AD pharmacotherapeutic strategies.

L-N-Acetylcysteine protects against radiation-induced apoptosis in a cochlear cell line

Conclusion. L-N-Acetylcysteine (L-NAC) significantly reduced reactive oxygen species (ROS) generation and cochlear cell apoptosis after irradiation. The safe and effective use of L-NAC in reducing radiation-induced sensorineural hearing loss (SNHL) should be verified by further in vivo studies. Objectives. Radiation-induced SNHL is a common complication after radiotherapy of head and neck tumours. There is growing evidence to suggest that ROS play an important role in apoptotic cochlear cell death from ototoxicity, resulting in SNHL. The aim of this study was to evaluate the effectiveness of L-NAC, an antioxidant, on radiation-induced apoptosis in cochlear cells. Materials and methods. The OC-k3 cochlear cell line was studied after 0 and 20 Gy of γ-irradiation. Cell viability assay was performed using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. Flow cytometry and TUNEL assay were done with and without the addition of 10 mmol/L of L-NAC. Intracellular generation of ROS was detected by 2′,7′-dichlorofluorescein diacetate, with comparisons made using fluorescence intensity. Results. L-NAC increased the viability of cells after irradiation. Generation of ROS was demonstrated at 1 h post-irradiation and was significantly reduced by L-NAC (p<0.0001). Flow cytometry and TUNEL assay showed cell apoptosis at 72 h post-irradiation, which was diminished by the addition of L-NAC.

Expression of a Functional Asialoglycoprotein Receptor in Human Renal Proximal Tubular Epithelial Cells

Background: The asialoglycoprotein receptor (ASGPR) is a C lectin which binds and endocytoses serum glycoproteins. In humans, the ASGPR is shown mainly to occur in hepatocytes, but does occur extrahepatically in thyroid, in small and large intestines, and in the testis. In the kidney, there has been evidence both for and against its existence in mesangial cells. Methods: Standard light microscopy examination of renal tissue stained with an antibody against the ASGPR was performed. The mRNA expression for the ASGPR H1 and H2 subunits in primary human renal proximal tubular epithelial cells (RPTEC), in the human proximal tubular epithelial cell line HK2, and in human renal cortex was investigated using reverse-transcribed nested polymerase chain reaction. ASGPR protein expression as well as ligand binding and uptake were also examined using confocal microscopy and flow cytometry (fluorescence-activated cell sorting). Results: Light microscopy of paraffin renal biopsy sections stained with a polyclonal antibody against the ASGPR showed proximal tubular epithelial cell staining of the cytoplasm and particularly in the basolateral region. Renal cortex and RPTEC specifically have mRNA for both H1 and H2 subunits of the ASGPR, but HK2 only expresses mRNA for H1. Using a monoclonal antibody, the presence of the ASGPR in RPTEC was shown by fluorescence-activated cell sorting and immunofluorescent staining. Specific binding and uptake of fluorescein isothiocyanate labelled asialofetuin which is a specific ASGPR ligand was also demonstrated in RPTEC. Conclusions: Primary renal proximal tubular epithelial cells have a functional ASGPR, consisting of the H1 and H2 subunits, that is capable of specific ligand binding and uptake.

Upregulation of AMPA receptor GluR2 (GluA2) subunits in subcortical ischemic vascular dementia is repressed in the presence of Alzheimer's disease

Glutamatergic AMPA receptors are of clinical significance in dementia because of their roles in mediating fast excitatory neurotransmission and other synaptic events relevant to cognition. Reductions in the AMPA receptor GluR2 subunit are well-established in Alzheimers disease (AD), but the status of GluR2 in subcortical ischemic vascular dementia (SIVD) and mixed AD/SIVD (MIX) has not been investigated. In this study we measured GluR2 immunoreactivity and mRNA levels in the postmortem neocortex of a longitudinally assessed cohort of SIVD and MIX, together with age-matched controls. We found that GluR2 immunoreactivity and mRNA were up-regulated in SIVD, but remained unchanged in MIX. Furthermore, higher GluR2 immunoreactivity was associated with milder cognitive impairment and lower concentrations of Aβ42 peptide and phosphorylated tau. Our study suggests that GluR2 up-regulation may be an adaptive process in SIVD, and that this process is repressed in the presence of concomitant AD in mixed dementia.

Genome-wide profiling of alternative splicing in Alzheimer's disease

Alternative splicing is a highly regulated process which generates transcriptome and proteome diversity through the skipping or inclusion of exons within gene loci. Identification of aberrant alternative splicing associated with human diseases has become feasible with the development of new genomic technologies and powerful bioinformatics. We have previously reported genome-wide gene alterations in the neocortex of a well-characterized cohort of Alzheimers disease (AD) patients and matched elderly controls using a commercial exon microarray platform [1]. Here, we provide detailed description of analyses aimed at identifying differential alternative splicing events associated with AD.

An isoform‐specific role of FynT tyrosine kinase in Alzheimer's disease

Alzheimers disease (AD) is the leading cause of dementia in old age and is characterized by the accumulation of β‐amyloid plaques and neurofibrillary tangles (NFT). Recent studies suggest that Fyn tyrosine kinase forms part of a toxic triad with β‐amyloid and tau in the disease process. However, it is not known whether Fyn is associated with the pathological features of AD in an isoform‐specific manner. In this study, we identified selective up‐regulation of the alternative‐spliced FynT isoform with no change in FynB in the AD neocortex. Furthermore, gene ontology term enrichment analyses and cell type‐specific localization of FynT immunoreactivity suggest that FynT up‐regulation was associated with neurofibrillary degeneration and reactive astrogliosis. Interestingly, significantly increased FynT in NFT‐bearing neurons was concomitant to decreased FynB immunoreactivity, suggesting an involvement of alternative splicing in NFT formation. Furthermore, cultured cells of astrocytic origin have higher FynT to FynB ratio compared to those of neuronal origin. Lastly, primary rat mixed neuron‐astrocyte cultures treated with Aβ25‐35 showed selective up‐regulation of FynT expression in activated astrocytes. Our findings point to an isoform‐specific role of FynT in modulating neurofibrillary degeneration and reactive astrogliosis in AD.

Decreased rabphilin 3A immunoreactivity in Alzheimer's disease is associated with Aβ burden

Synaptic dysfunction, together with neuritic plaques, neurofibrillary tangles and cholinergic neuron loss is an established finding in the Alzheimers disease (AD) neocortex. The synaptopathology of AD is known to involve both pre- and postsynaptic components. However, the status of rabphilin 3A (RPH3A), which interacts with the SNARE complex and regulates synaptic vesicle exocytosis and Ca(2+)-triggered neurotransmitter release, is at present unclear. In this study, we measured RPH3A and its ligand Rab3A as well as several SNARE proteins in postmortem neocortex of patients with AD, and found specific reductions of RPH3A immunoreactivity compared with aged controls. RPH3A loss correlated with dementia severity, cholinergic deafferentation, and increased β-amyloid (Aβ) concentrations. Furthermore, RPH3A expression is selectively downregulated in cultured neurons treated with Aβ25-35 peptides. Our data suggest that presynaptic SNARE dysfunction forms part of the synaptopathology of AD.

Selective induction of alternatively spliced FynT isoform by TNF facilitates persistent inflammatory responses in astrocytes

Fyn tyrosine kinase has been implicated in the pathogenesis of Alzheimer’s disease (AD). We have previously reported that upregulation of the FynT isoform in AD brains was partly associated with astrocyte activation. In this study, we demonstrated selective FynT induction in murine cortex and primary astrocyte culture after prolonged exposure to inflammatory stimulants, suggesting that FynT may mediate persistent neuroinflammation. To delineate the functional role of astrocytic FynT in association with TNF-mediated inflammatory responses, immortalized normal human astrocytes (iNHA) stably expressing FynT kinase constitutively active (FynT-CA) or kinase dead (FynT-KD) mutants were treated with TNF and compared for inflammatory responses using high-throughput real-time RT-PCR and Luminex multi-analyte immunoassays. FynT-CA but not FynT-KD mutant exhibited drastic induction of proinflammatory cytokines and chemokines after prolonged exposure to TNF, which could be attenuated by treating with Fyn kinase inhibitor PP2 or silencing via FynT-specific DsiRNA. FynT kinase activity-dependent induction of PKCδ expression, PKCδ phosphorylation, as well as NFκB activation was detected at the late phase but not the early phase of TNF signaling. In conclusion, selective FynT induction by TNF may facilitate persistent inflammatory responses in astrocytes, which is highly relevant to chronic neuroinflammation in neurodegenerative diseases including but not limited to AD.

A Randomised Controlled Trial Evaluating the Efficacy of a Nurse Controlled Analgesia (NCA) Protocol in Post Anaesthesia Care Unit (PACU)

Introduction: The purpose of this trial was to determine the efficacy of a Nurse Controlled Analgesia (NCA) protocol in reducing the time taken to achieve a satisfactory postoperative pain score. The safety aspects of the NCA protocol were also evaluated. Method: 120 patients undergoing major surgery were randomised into 2 groups – NCA group and the doctor administered analgesia group (DRA). Nurses in the Post Anaesthestic Care Area (PACU) administered analgesia based on a protocol whereas analgesia was titrated at the discretion of the doctor in the DRA group. Effective pain control was measured by the time taken to obtain first dose of analgesia and the time taken to achieve either a pain score of less than 4/10 or if the patient refused further analgesia. Results: Patients received their first dose of morphine faster in the NCA group compared to DRA group, 6.7 minutes versus 9.5 minutes respectively (p= 0.037). Time taken to achieve a pain score of less than 4 was shorter in the doctor group although this was not significant. Complication rates were not different in both groups. Conclusion: Nurse administered analgesia based on a protocol can provide pain relief for patients in the postoperative period as effectively and safely as doctor administered analgesia.